We initially focused on a PIV3-based vector to express RSV F protein, given the pediatric impact of both viruses. We used an attenuated HPIV3 vaccine candidate called B/HPIV3 that we previously developed. This virus consists of bovine PIV3 in which the F and HN genes have been replaced by those of HPIV3, yielding a chimeric virus that is attenuated in primates due to the bovine backbone, and which bears the neutralization and major protective F and HN antigens of HPIV3. Both the empty B/HPIV3 vector and B/HPIV3 expressing the unmodified RSV F protein were previously shown to be well-tolerated in infants and young children. Therefore, the crucial factor of safety has already been demonstrated for this vector. A number of strategies were evaluated previously to optimize the immunogenicity of the expressed RSV F protein. Evaluation of several different positions for the RSV F gene in the vector genome identified the second gene position as generally being optimal. Evaluation of several versions of codon-optimization of the RSV F ORF identified the most efficient sequence. The RSV F protein was modified to be identical to an early-passage isolate, which reduced fusion and stabilized the trimer. We also engineered RSV F to be efficiently packaged in the B/HPIV3 vector particle. This was done by replacing the transmembrane domain and cytoplasmic tail (TMCT) of RSV F with those of BPIV3 F. This modification was substantially attenuating but resulted in a large increase in the titers of serum RSV-neutralizing antibodies when assayed in the absence of complement, an assay that detects strongly-neutralizing antibodies. We sought to improve the quality of the RSV F antigen by increasing the stability of the prefusion conformation (the conformation that is the most efficient in inducing neutralizing antibodies) by structure-based engineering, using mutations that have been reported by colleagues in the NIH Vaccine Research Center and elsewhere. This included the addition of a disulfide bond (called the DS mutation) on its own and in combination with two cavity-filling mutations (called Cav1). Both the DS and DS-Cav1 forms of RSV F resulted in a large increase in serum RSV-neutralizing antibodies that neutralized efficiently in vitro without added complement. The combination of DS-Cav1 prefusion stabilization, optimized codon-usage, and vector packaging significantly improved vector immunogenicity and protective efficacy against RSV. This provides an improved PIV3-vectored RSV vaccine candidate that will be manufactured into clinical trial maternal for pediatric clinical evaluation. During the present report period, we constructed and evaluated B/HPIV3 constructs expressing additional versions of stabilized prefusion RSV F protein, but these studies are still in progress. We also are constructing and evaluating versions of PIV3 vectors that are less attenuated on their own than B/HPIV3. The use of such a vector would accommodate the added attenuation that results from packaging of the TMCT versions of RSV F. We also used the B/HPIV3 vector to express the RSV attachment glycoprotein (G), which is the second RSV neutralization antigen and a major protective antigen. G contains a conserved fractalkine-like CX3C motif and is expressed as membrane-anchored (mG) and secreted (sG) forms. The CX3C motif and sG are widely thought to interfere with host immune responses, and it has been suggested that elimination of these features would improve the immunogenicity of an RSV vaccine. We used the rB/HPIV3 vector to express wt RSV G and various modified forms, including sG, mG, mutants with ablated CX3C motif, and mutants bearing the TMCT of BPIV3 HN to achieve enhanced packaging into vector virions. Using a hamster model, we evaluated the effects of these individual factors on immunogenicity and protective efficacy induced against both the vector and the RSV insert. Furthermore, since PIV3 and RSV have similar tropisms and patterns of replication in vivo, evaluation of the replication of the B/HPIV3 vector provided a means to detect possible changes in the pulmonary immune milieu that might affect RSV/PIV3 replication. In hamsters, all vector constructs replicated to similar titers in the upper and lower respiratory tracts, allowing direct comparison of immune responses. Ablation of sG did not affect the RSV- or PIV3-neutralizing antibody (NAb) responses. Increased packaging did not affect the immunogenicity of RSV G, in contrast to previous findings with RSV F. Mutation of the CX3C motif drastically reduced the G-specific serum NAb response and protection against RSV challenge, indicating the importance of the integrity of the CX3C motif for the immunogenicity of RSV G. In human airway epithelium (HAE) cultures, serum NAbs induced by wt RSV G , but not by wt RSV F, completely blocked RSV infection in the absence of added complement. This activity was reduced if the CX3C motif in the G immunogen was ablated. This suggests that NAbs induced by wt RSV G conferred more complete protection of the epithelium than NAbs induced by wt RSV F. In addition, vector expressing wt G was more protective in hamsters than that expressing wt F against RSV challenge. Increased expression of wt RSV G by codon-optimization increased the immunogenicity and protective efficacy. This study showed that ablation of the CX3C motif in an RSV vaccine, as has been suggested by some, would be ill-advised, and ablation of sG would have no benefit. It suggests that RSV G would be an important component of an RSV vaccine. We also extended the principles described above to HPIV1 vectors. We previously compared HPIV1 vectors that were attenuated by different mutations, and selected a vector containing a 6-nt deletion in the P/C gene (called C-del170), that reduces the ability of HPIV1 to block host interferon and apoptosis responses. We evaluated RSV F protein modified for increased stability in the prefusion conformation by the previously-described DS-Cav1 mutations. RSV F was expressed from the first or second gene position. The TMCT domains were substituted with those of HPIV1 F, resulting in efficient packaging into the HPIV1 vector virions. In hamsters, the presence of the RSV F gene, and in particular the TMCT versions, was substantially attenuating and thereby resulted in reduced immunogenicity (due to reduced antigen expression). However, the vector expressing full-length RSV F from the pre-N position was immunogenic for RSV and HPIV1. It conferred complement-independent high-quality RSV-neutralizing antibodies at titers similar to those of wild type RSV and provided protection against RSV challenge. The vectors exhibited stable RSV F expression in vitro and in vivo. In conclusion, an attenuated rHPIV1 vector expressing a pre-F-stabilized form of RSV F demonstrated promising immunogenicity and should be further developed as an intranasal bivalent pediatric vaccine.